I.
Surgical
occlusion
of
the
distal
medial
cerebral
artery
(MCA)
to
induce
ischemia
a.
Mice
are
anesthetized
intraperitoneally
(i.p.)
with
cocktail
combination
of
ketamine
(100
mg/kg)
and
xylazine
(5
mg/kg).
b.
Anesthetized
mice
are
placed
on
an
operating
table
with
a
heating
pad
maintained
at
37°C
and
under
a
dissecting
stereomicroscope.c.
While
adhering
to
aseptic
surgical
techniques,
a
0.5
cm
vertical
skin
incision
is
made
midway
between
the
lateral
part
of
the
orbit
and
the
external
auditory
meatus
(Fig.
1).
d.
The
The
rostral
pole
of
the
parotid
gland
and
the
upper
part
of
the
temporal
muscle
are
pushed
aside
after
partial
resection
to
expose
the
cranium.
e.
A
small
craniotomy
is
then
made
with
a
0.2-mm
burr
at
the
junction
of
the
zygomatic
arch
and
the
squomosal
bone,
and
above
the
anterior
distal
branch
of
the
MCA,
using
a
high-speed
microdrill
(Fig.
1).f.
The
sectioned
layer
of
the
skull
is
removed
with
fine
forceps,
and
the
dura
and
arachnoid
layers
are
carefully
opened
to
expose
the
brain
superficial
vasculature
(Fig.
2).g.
The
right
MCA
distal
to
the
lenticulostriate
branches
is
localized
using
a
stereomicroscope,
and
electrocauterized
using
a
microcauterizer
(Fig.
2).
h.
The
coagulated
MCA
segment
is
then
transected
with
Vannas
scissors
to
verify
that
the
occlusion
is
permanent.i.
The
muscle
and
soft
tissue
are
replaced
and
the
surgical
site
is
closed
with
6-0
sterile
nylon
sutures.j.
4%
lidocaine
cream
is
applied
over
the
closed
surgical
site
as
a
local
anesthetic,
administered
1
mL
of
physiologic
0.9%
saline
s.q.
to
prevent
dehydration,
and
eyes
coated
with
artificial
tears
to
protect
from
drying.
k.
Mice
are
maintained
at
37°C
after
surgery
until
fully
recovered
from
anesthesia.
l.
Fully
recovered
mice
are
allowed
free
access
to
food
and
water
in
their
cages
for
24
h.

Figure
2.
Schematic
illustration
and
landmarks
of
the
right
middle
cerebral
artery
(MCA)
(Panels
A
and
B).
Outline
of
cerebral
vasculature
(Panel
C).

II.
Brain
tissue
processing
a.
Twenty-four
hours
after
surgery,
the
mice
are
euthanized
by
cervical
dislocation.b.
The
brain
is
carefully
dissected
and
removed
en
bloc
from
the
calvarium
and
placed
in
a
previously
cooled
matrix
for
sectioning.c.
1
mm
coronal
sections
of
the
brain
are
made
while
contained
within
the
matrix
(Fig.
3,
left
panel).d.
Brain
slices
are
placed
in
12
well
plates,
freed
from
the
dura
mater
and
vascular
tissue,
and
then
soaked
in
a
solution
of
2%
TTC
(2,3,5
-triphenyltetrazolium
chloride)
in
0.1
mol/L
PBS
(pH
7.4).e.
Brain
slices
are
incubated
in
a
37°C
water
bath
for
10-30
min
with
gentle
shaking
to
ensure
even
exposure
of
the
surfaces
to
staining.
f.
Excess
TTC
is
then
drained
and
brain
slices
are
refrigerated
in
10%
phosphate-buffered
formalin.g.
The
sections
are
stored
in
this
manner
for
up
to
seven
days
until
the
tissues
are
scanned
for
further
analysis.

III.
Infarct
volume
morphometry
using
image
analysisa.
All
image
collection,
processing
and
analysis
are
performed
in
a
blind
manner
and
under
controlled
environmental
lighting.
b.
Each
brain
slice
is
placed
with
its
frontal
surface
toward
the
scanner.
(The
most
anterior
and
posterior
sections
are
not
used
because
of
variability
in
size.)c.
All
sections
in
the
12
well
plates
are
scanned
simultaneously.
The
lid
is
closed
and
black
felt
covered
the
plate
for
greater
contrast.
d.
The
scanner
settings
to
acquire
the
images
are:
300
dots
per
inch
(dpi),
millions
of
colors,
auto
contrast
off
and
no
active
adjustment.
e.
Acquisition
of
the
image
is
done
through
Adobe
Photoshop
and
with
an
Apple
compatible
scanner
within
the
Adobe
Photoshop
Scanwizard
plugin.f.
The
raw
files
are
then
saved
as
uncompressed
TIFF
files
(tagged
image
file
format),
written
onto
a
CD-R
and
further
backed
up
onto
a
server.g.
Images
are
then
analyzed
using
image
analysis
software
(i.e.
SPOT).
Magic
Wand
tool
is
used
to
autotrace
the
specific
areas
of
interest.h.
Infarct
volumes
are
calculated
by
measuring
infarct
areas
on
the
separate
slices,
multiplying
areas
by
slice
thickness,
and
summing
all
slices;
this
“indirect”
morphometric
method
corrects
for
edematous
swelling.

Definitions
&
calculations

Craniotomy:
is
a
surgical
procedure
where
in
a
piece
of
bone
is
temporarily
removed
from
the
cranium
or
skull
providing
access
to
the
brain.